Mechanical Force and Notch Signaling

Abstract

The mechanical microenvironment of a cell guides cellular processes such as migration, differentiation, division, and signaling, and is thus often altered in disease. At a molecular level, mechanical stimuli alter conformations of mechanosensing proteins to communicate the stimulus to the cell interior and effect a downstream cellular response in a process called mechanotransduction. Measuring tensions sensed by cellular proteins and how tensions are altered by cellular context will lead to better understanding of the molecular mechanisms used in mechanosensing in disease‐related processes such as tumor migration. Tools to measure molecular level tensions have only emerged more recently to characterize the pN forces present across mechanosensitive proteins and have been used to measure tensions sensed by focal adhesions proteins such as vinculin, talin, and integrins, as well as cadherins. Generally, genetically encodable molecular tension sensors contain a deformable peptide linker with well‐characterized extension in response to applied force flanked by two fluorescent proteins (FP). The extent of linker stretching due to mechanical force can measured by Förster resonance energy transfer (FRET) and subsequently be correlated to a quantifiable force. We developed a genetically‐encodable molecular tension sensor based on bioluminescence resonance energy transfer (BRET) that overcomes several limitations of FRET. Using BRET as a distance reporter in molecular tension sensors allows for more sensitive readouts due to the lack of autofluorescence and phototoxicity. BRET also offers the potential for use in vivo where light excitation is not able to penetrate into tissue. We demonstrate the utility of this tension sensor in vitro and in cell based assays, using vinculin as the model system to show an enhanced dynamic range compared to field standard FRET‐based sensors. We will then present measurements of tensions sensed between cells by Notch receptors.Support or Funding InformationPew Biomedical Scholar, R35 GM119483